biochemistry 201 biological regulatory mechanisms transcription and its regulation january 22...
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Biochemistry 201Biological Regulatory Mechanisms
Transcription and Its Regulation
January 22 –Mechanism of Transcription InitiationJanuary 24– Mechanism of Transcription ElongationJanuary 28– Control of Transcription in BacteriaJanuary 31– Control of Transcription in Eukaryotes
Mechanism of Transcription Initiation
ReferencesI. General
Chapter 12 of Molecular Biology of the Gene 6 th Edition (2008) by Watson, JD, Baker, TA, Bell, SP, Gann, A, Levine, M, Losick, R. 377-4142.2. ReviewsMurakami KS, Darst SA. (2003) Bacterial RNA polymerases: the wholo story. Curr Opin Struct Biol 13:31-9.
Campbell, E, Westblade, L, Darst, S., (2008) Regulation of bacterial RNA polymerase factor activity: a structural perspective. Current Opinion in Micro. 11:121-127
Herbert, KM, Greenleaf, WJ, Block, S. (2008) Single-Molecule studies of RNA polymerase: Motoring Along. Annu Rev Biochem. 77:149-76.
Werner, Finn and Dina Grohmann (201). Evolution of multisubunit RNA polymerases in the three domains of life. Nature Rev. Microbiology 9: 85-98
3. Studies of Transcription InitiationRoy S, Lim HM, Liu M, Adhya S. (2004) Asynchronous basepair openings in transcription initiation: CRP enhances the rate-limiting step. EMBO J. 23:869-75.
Sorenson MK, Darst SA. (2006).Disulfide cross-linking indicates that FlgM-bound and free sigma28 adopt similar conformations. Proc Natl Acad Sci U S A. 103:16722-7.
Young BA, Gruber TM, Gross CA. (2004) Minimal machinery of RNA polymerase holoenzyme sufficient for promoter melting. Science. 303:1382-1384
*Kapanidis, AN, Margeat, E, Ho, SO,.Ebright, RH. (2006) Initial transcription by RNA polymerase proceeds through a DNA-scrunching mechanism. Science. 314:1144-1147.
Revyakin A, Liu C, Ebright RH, Strick TR (2006) Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching. Science. 314: 1139-43.
Murakami KS, Masuda S, Campbell EA, Muzzin O, Darst SA (2002). Structural basis of transcription initiation: an RNA polymerase holoenzyme-DNA complex. Science. 296:1285-90.
Kostrewa D, Zeller ME, Armache KJ, Seizl M, Leike K, Thomm M, Cramer P.(2009) RNA polymerase II-TFIIB structure and mechanism of transcription initiation. Nature. 462:323-30.
Discussion Paper**Feklistov A and Darst, SA (2011) Structural basis for Promoter -10 Element recognition by the Bacterial RNA Polymerase Subunit. Cell 147: 1257 – 1269Accompanying preview: Liu X, Bushnell DA and Kornberg RD ( 2011) Lock and Key to Transcription: –DNA Interaction. Cell: 147: 1218-1219
***Paul BJ, Barker MM, Ross W, Schneider DA, Webb C, Foster JW, Gourse RL. (2004) DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP.Cell. 6:311-22.
The accompanying minireview is helpfulNickels, B.E. and Hochschild, A. (2004) Regulation of RNA Polymerase through the Secondary Channel. Cell 118:281-284
Key Points1. Multisubunit RNA polymerases are conserved among all organisms
2. RNA polymerases cannot initiate transcription on their own. In bacteria 70 is required to initiate
transcription at most promoters. Among other functions, it recognizes the key features of most bacterial promoters, the -10 and -35 sequences.
2. E. coli RNA polymerase holoenzyme, (core + finds promoter sequences by sliding along DNA and by transfer from one DNA segment to another. This behavior greatly speeds up the search for specific DNA sequences in the cell and probably applies to all sequence-specific DNA-binding proteins.
3. Transcription initiation proceeds through a series of structural changes in RNA polymerase, 70 and DNA.
4. A key intermediate in E. coli transcription initiation is the open complex, in which the RNA polymerase holoenzyme is bound at the promoter and ~12 bp of DNA are unwound at the transcription startpoint. Open complex formation does not require nucleoside triphosphates. Its presence can be monitored by a variety of biochemical and structural techniques.
5. Recognition of the -10 element of the promoter DNA is coupled with strand separation
6. When the open complex is given NTPs, it begins the ‘abortive initiation’ phase, in which RNA chains of 5-10 nucleotides are continually synthesized and released.
7. Through a “DNA scrunching” mechanism the energy captured during synthesis of one of these short transcripts eventually breaks the enzyme loose from its tight connection to the promoter DNA, and it begins the elongation phase.
7. Aspects of the mechanism of initiation are likely to be conserved in eukaryotic RNA polymerase
rRNAs snRNAs miRNAs
Other non-coding RNAs (e.g. telomerase RNA)
mRNAs
translation
proteins
transcription
(RNA processing)
Transcription is Important
Transcription/Splicing/Translation ProvideA Large Range of Protein Concentrations
I. RNA polymerases
Cellular RNA polymerases in all living organisms are evolutionary related
a common structural and functional frame work of transcription in the three domains of life
LUCA-Last universal common ancestor
Sub
units
of R
NA
P
Structure of RNAP in the three domains Structure of RNAP in the three domains
Werner and Grohmann (2011),Nature Rev Micro 9:85-98
Extra RNAP subunits provide interaction sites for transcription factors, DNA and RNA, and modulate diverse RNAP activities
Universally conserved
Archaeal/eukaryotic
Bacteria Archaea Eukarya
Transcription
Evolutionary relationships of general transcription factorsEvolutionary relationships of general transcription factors
Initiation
GreTranscript cleavage
Elongation
LUCA may have had elongating, not initiating RNA polymerase
II. Challenges in initiating transcription
1. RNAP is specialized to ELONGATE, not INITIATE
2. Initiating RNAP must open DNA to permit transcription
3. RNAP must leave promoter—abortive initiation
The Initiating Form of RNA Polymerase
‘holoenzyme’
'
KD ~ 10-9 M
+
‘core’}
Can begin transcription on
promoters and can elongate
}Can elongate but
cannot begin transcription at
promoters
factor is required for bacterial RNA polymerase to initiate transcription on promoters
'
(1) The discovery of initiation factors
How was discovered (Burgess, 1969)
A. Assay for RNA polymerase:
E.coli lysate
buffer
*ATPCTPGTPUTP
Calf thymus DNA
Look for incorporation of *ATP into RNA chains
B. Initial purification
Lysate
various fractionation steps (DEAE column, glycerol gradient etc)
Active fractions identified by assay
Labmate Jeff Roberts reported that the new, improved preparation of RNAP (peak 2) had no activity on DNA
Peak 1 restored activity
C. Improved purification of RNA polymerase:
Improved fractionationlysate
phosphocellulose column
salt
OD
28
0
1
2
Act
ivit
y (
*ATP)
CT D
NA
Fraction #
SDS gel analysis Peak 1 Peak 2
'
increases rate of initiation
g
Tra
nsc
ripti
on
D
NA Assay:
incorporationP
ATP
(2) Bacterial promoters
There are several flavors of promoters
and recruit RNAP to promoter DNA
(3) undergoes a large conformational change upon binding to RNA polymerase
Free doesn’t bind DNA in holoenzyme positioned for DNA recognition Sorenson; 2006
is positioned for DNA recognition
is positioned to affect key activities of RNA polymerase
Surprising structural similarity between the initiating
forms of bacterial and eukaryotic RNAP
The first two steps of Eukaryotic transcription
Many archae have a proliferation of TBPs and TFBs, suggesting that they provide choice in promoters, akin to alternative s.
In archae, TBP and TFB are sufficient for formation of the pre-initiation complex (PIC), suggesting that they are key to the mechanism of transcription initiation in eukaryotes
Promoter
TFBTBP
D Kostrewa et al. Nature 462, 323-330 (2009) doi:10.1038/nature08548
TFIIB has a central role in initiation similar to that of
Recruits Pol II to promoter: N-terminus binds Pol II; C terminus binds TBP and DNA
TFIIB structure
Role in promoter opening; B linker mutants recruit PolII but cant strand open or initiate
Role in selection of TSS ( Inr): B reader mutants
Blocks elongating RNA chain: B reader
Crystal structure of TFB + RNA polymerase--archae
Topological similarities in /TFIIB binding to RNAP
B reader ( 3.2): both in exit channel and near active site; start site selection
B ribbon (4):both bind flap tip helix
B linker (2): both bind coiled -coil and rudder; both involved in strand opening
B core (3)
D Kostrewa et al. Nature 462, 323-330 (2009) doi:10.1038/nature08548
TFIIB and bound to RNA polymerase show surprising similarity. Analogously placed regions have similar functions
Initiating RNAP must open DNA to permit transcription:Formation of the open complex
Steps in transcription initiation
KB Kf
initial binding
“isomerization”
Abortive Initiation
ElongatingComplex RPoRPcR+P
NTPs
A detailed look at a prokaryotic promoter
Sequence Logos-35 logo -10 logo
TT TT AAGG CC AA TT AA TT AA AA TT15-
19nucleotides
Is the -10 promoter element recognized as Duplex or SS DNA?
-10 logo-35 logo
Helix-turn-helix in Domain 4Recognizes -35 as duplex DNA
Recognition of the prokaryotic promoter
Approach
1. Determine a high resolution structure of 2 bound to non-template strand of the -10 element
2. Determine whether this structure represents the “initial binding state” or endpoint state
Schematic
Promoter escape
is positioned to affect key activities of RNA polymerase
Promoter escape and Abortive Initiation
during abortive initiation, RNAP synthesizes many short transcripts, but reinitiates rapidly. How can the active site of RNAP move forward
along the DNA while maintaining promoter contact?
Förster (fluorescence) resonance energy transfer (FRET) allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. When the donor (yellow star) is excited (blue arrows) it emits light. When the donor fluorophore moves sufficiently close to the acceptor (right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.This process depends on the inverse sixth power of the distance between fluorophores. By measuring the intensity change in acceptor fluorescence, distances on the order of nanometers can currently be measured in single molecules with millisecond time resolution
Experimental set-up for single molecule FRET: Single transcription complexes labeled with a fluorescent donor (D, green) and a fluorescent acceptor (A, red) are illuminated as they diffuse through a femtoliter-scale observation volume (green oval; transit time ~1 ms); observed in confocal microscope
Using single molecule FRET to monitor movement of RNAP and DNA
Three models for Abortive initiation
#1
Predicts expansion and contraction of RNAP
Predicts expansion and contraction of DNA
Predicts movement of both the RNAP leading and trailing edge relative to DNA
#2
#3
A. N. Kapanidis et al., Science 314, 1144 -1147 (2006)
Initial transcription involves DNA scrunching
Lower E* peak is free DNA; higher E* peak is DNA in open complex; distance is shorter because RNAP induces DNA bending
Open complex
Initial transcription involves DNA scrunching
Higher E* in Abortive initiation complex than open complex results from DNA scrunching
Open complex
Abortive initiation complex
Initial transcription involves DNA scrunching
Open complex
Abortive initiation complex
At a typical promoter, promoter escape occurs only after synthesis of an RNA product ~9 to 11 nt in length (1–11) and thus can be inferred to require scrunching of ~7 to 9 bp (N – 2, where N = ~9 to 11; Fig. 3C). Assuming an energetic cost of base-pair breakage of ~2 kcal/mol per bp (30), it can be inferred that, at a typical promoter, a total of ~14 to 18 kcal/mol of base-pair–breakage energy is accumulated in the stressed intermediate. This free energy is high relative to the free energies for RNAP-promoter interaction [~7 to 9 kcal/mol for sequence-specific component of RNAP-promoter interaction (1)] and RNAP-initiation-factor interaction [~13 kcal/mol for transcription initiation factor {sigma}70 (31)].
The energy accumulated in the DNA scrunched “stressed intermediate could disrupt interactions between RNAP,
and the promoter, thereby driving the transition from initiation to elongation
Validation of the prediction that occlusion of the RNA exit channel promotes “abortive initiation”
#1: transcription by holoenzyme with full-length #2: transcription by holoenzyme with truncated at Region 3.2: lacks in the RNA exit channel
Murakami, Darst 2002
is positioned to affect key activities of RNA polymerase